Endochondral bone formation consists of a developmental cascade of cellular differentiation that culminates in extracellular matrix mineralization. The process is required for normal growth and development of long bones and for certain kinds of bone repair. During the chondrogenic phase of the process, chondrocytes are responsible for the synthesis, maintenance and maturation of a calcifiable extracellular matrix that is composed mainly of proteoglycan and collagen. (Boskey, A. L. (1991), "Current concepts of the physiology and biochemistry of calcification," Clin. Orthop. 157:225-257; Howell, D. S. and Dean, D. D. (1992), "Biology, chemistry and biochemistry of the mammalian growth plate," In: Disorders of Bone and Mineral Metabolism, Coe, F. L. and Favus, M. J. (eds), Raven Press Ltd., New York 313-353.)
The complex regulation of chondrocyte differentiation by growth factors such as TGF.beta. and other hormones has been shown by numerous investigators. (Crabb, I. D., et al. (1990), "Synergistic effect of transforming growth factor-.beta. and fibroblast growth factor on DNA synthesis in chick growth plate chondrocytes," J. Bone Min. Res. 5:1105-1112; Kinoshita, A., et al. (1992), "Demonstration of receptors for epidermal growth factor on cultured rabbit chondrocytes and regulation of their expression by various growth and differentiation factors," Biochem. Biophys. Res. Comm. 183:14-20; Suzuki, F. (1992), "Effects of various growth factors on a chondrocyte differentiation model," Adv. Exper. Med. and Biol. 324:101-106; Thorp, B. H., et al. (1992), "Transforming growth factor-.beta.1, -.beta.2, and -.beta.3 in cartilage and bone cells during endochondral ossification in the chick," Development 114:907-911).
Vitamin D.sub.3 is known to be an essential regulator of this complex process, and both 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 are involved. (Raisz, L. G. and Kream, B. E. (1983), "Regulation of bone formation," (first of two parts), N. Engl. J. Med. 309:29-35; Raisz, L. G. and Kream, B. E. (1983), "Regulation of bone formation," (second of two parts), N. Engl. J. Med. 309:83-89; Canterbury, J. M., et al. (1980), "Metabolic Consequences of oral administration of 24,25 hydroxycholecalciferol to uremic dogs," J. Clin. Invest. 65:571-580; Liberherr, M. et al. (1979), "Interaction of 24,25-dihydroxyvitamin D.sub.3 and parathyroid hormone on bone enzymes in vitro," Calcif. Tissue Int. 27:47-53; Ornoy, A., et al. (1978), "24,25-Dihydroxyvitamin D.sub.3 is a metabolite of vitamin D essential for bone formation," Nature 276:517-520; and Norman, A. W. (1980), "1,25-Dihydroxyvitamin D.sub.3 and 24,25-dihydroxyvitamin D.sub.3 : key components of the vitamin D endocrine system. Contr. Nephrol. 18:1-11; Grigoriadis, A. E., et al. (1989), "Effects of dexamethasone and vitamin D.sub.3 on cartilage differentiation in a clonal chondrogenic cell population," Endocrinology 125:2103-2110; Schwartz, Z., et al. (1992), "Direct effects of transforming growth factor .beta. on chondrocytes are modulated by vitamin D metabolites in a cell maturation specific manner," Endocrinology 132:1544-1552; Schwartz, Z. et al., "Differential Regulation of prostaglandin E2 synthesis and phospholipase A.sub.2 activity by 1,25-(OH).sub.2 D.sub.3 in three osteoblast-like cell lines (MC-373-E1), ROS 17/2.8 and MG-63", Bone (1992) 13:51-58.)
Matrix vesicles, and the phospholipids present in them, are involved in initial formation of calcium hydroxyapatite crystals via the interaction of calcium and phosphate ions with phosphatidylserine to form phospholipid:Ca:Pi complexes (CPLX). CPLX is present in tissues which are undergoing initial mineral deposition but are absent from nonmineralizing tissues. Evidence suggests that CPLX resides in the interior of matrix vesicles where the earliest mineral crystals are formed in association with the vesicle membrane. More recently, it has been determined that specific membrane proteins, called proteolipids, participate in CPLX formation and hydroxyapatite deposition, in part by structuring phosphatidylserine in an appropriate conformation. Phosphatidylserine involvement in the initiation of mineralization has been extensively investigated because of its extremely high binding affinity for Ca.sub.2+. In addition to structuring a specific phospholipid environment, proteolipids may also act as ionophores, promoting export of protons and import of calcium and phosphate, both requirements of biologic calcification (Boyan, B. D. et al., "Role of lipids in calcification of cartilage," Anat. Rec. (June 1989) 224(2):211-219).
There is a known correlation between in vivo bone formation and in vitro production of normal matrix vesicles (Boyan, B. D. et al., "Epithelial cell lines that induce bone formation in vivo produce alkaline phosphatase-enriched matrix vesicles in culture," Clin. Orthop. (April 1992) 266-276).
Many cells produce growth factors in latent form and store them in their extracellular matrix, or they may store them in an inactive form via specific binding proteins. These growth factors may be activated at a later time and act on the original cell as autocrine factors, or a neighboring cell as paracrine factors, or they may be released into the circulation and have a systemic effect as endocrine agents. One function of the extracellular matrix vesicles is to transport enzymes for matrix modification (Boskey, A. L. et al., "Studies of matrix-vesicle-induced mineralization in a gelatin gel," Bone Miner. 17:257-262). Matrix vesicles are selectively enriched in enzymes that degrade proteoglycans (Dean, D. D. et al., "Matrix vesicles contain metaloproteinases that degrade proteoglycans," Bone Miner. (1992) 17:172-176).
Transforming growth factor beta (TGF.beta.) is an important regulator of cartilage development and chondrocyte differentiation (Seyedin, S. M., et al., J. Biol. Chem (1987) 262:1946-1949; Seyedin, S. M., et al., Proc. Natl. Acad. Sci. USA (1985) 82:2267-2271; Seyedin, S. M., et al., J. Biol. Chem. (1986) 261:5693-5695). It is synthesized by chondrocytes and appears to act in an autocrine manner (Gelb, D. E., et al., Endocrinology (1990) 127:1941-1947; Schwartz, Z., et al., "Direct effects of transforming growth factor-beta on chondrocytes are modulated by vitamin D metabolites in a cell maturation-specific manner," Endocrinology (1993) 132:1544-1552; Rosier, R. N., et al., Connect. Tissue Res. (1989) 20:295-301). TGF.beta. production varies with stage of chondrocyte differentiation.
TGF.beta. is produced by many cell types in a latent form which may be released into the circulation, as during platelet lysis (Wakefield, L. M., et al., J. Biol. Chem. (1988) 263:7646-7654; Miyazono, K., et al., J. Biol. Chem. (1988) 263:6407-6415) or targeted for storage in the extracellular matrix (Dallas, S. L., et al., J. Biol. Chem. (1994) 269:6815-6822). Latent TGF.beta. exists in a number of macromolecular forms. Recombinant human TGF.beta..sub.1 is a homodimer of 100 kD which contains a latency-associated peptide non-covalently bound to the mature TGF.beta. molecule (Gentry, L. E., et al. (1987), Mol. Cell. Biol. 7:3418-3427). Latent TGF.beta. synthesized by fibroblasts consists of a similar or identical 100 kD homodimer covalently bound through a cysteine residue to a 190 kD TGF.beta. binding protein (Kanzaki, T., et al. (1990), Cell 61:1051-1061; Tsujmi, T., et al. (1990), Proc. Natl. Acad. Sci. U.S.A. 87:8835-8839). Platelets produce a latent TGF.beta. that contains a truncated form of the 190 kD binding protein (Wakefield, et al. (1988), J. Biol. Chem. 263:7646-7654). Bone cells produce large amounts of the 100 kD complex (Bonewald, L. et al. (1991), Mol. Endocrinol. 5:741-751) in addition to the fibroblast form of latent TGF.beta. (Dallas, S. L., et al. (1994), J. Biol. Chem. 269:6815-6822).
Storage of latent TGF.beta. and the mechanism, as well as timing, of activation of latent TGF.beta. appears to be specific for each cell and tissue type. A variety of factors may stimulate cells to activate latent TGF.beta.. For example, macrophages treated with .gamma.-interferon activate latent TGF.beta. (Twardzik, D. R., et al., Ann. N.Y. Acad. Sci. (1990) 593:276-284), as will osteoclasts treated with retinol (Oreffo, R.O.C., et al., Biochem. Biophys. Res. Comm. (1989) 153:817-823).
Local production of acid may be one mechanism by which latent TGF.beta. is activated. For example, it is believed that latent TGF.beta. in milk is activated by stomach acid and that the active form is transported through the gut (Saito, S., et al., Clin. Exp. Immunol. (1993) 94:220-224). While acid pH can activate latent TGF.beta., it is clear that proteases play an important role in most systems. Endothelial cells activate latent TGF.beta. through the plasmin system (Sato, Y. and Rifkin, D. B., J. Cell Biol. (1989) 109:309-315). Arian osteoclasts appear to use multiple proteases in addition to acid pH (Oursler, M. J., J. Bone Min. Res. (1994) 9:443-452). In growth plate cartilage and unmineralized osteoid in bone, where local generation of acid has not been reported, participation of proteases is an attractive option.
Recent studies have shown that proteinases, including neutral and acid metalloproteinases and plasminogen activator, and various peptidases are present at high levels in matrix vesicles (Hirschman, A., et al., Calcif. Tissue Int. (1983) 35:791-797; Einhorn, T. A., et al., J. Orthop. Res. (1989) 7:792-805; Dean, D. D., et al., "Matrix vesicles are enriched in metalloproteinases that degrade proteoglycans," Calcif. Tissue Int. (1992) 50:342-349). These extracellular organelles are membrane bounded, produced by chondrocytes and osteoblasts in vivo (Anderson, H. C., J. Cell Biol. (1969) 41:59-72; Schwartz, Z., et al., Bone (1989) 10:53-60) and in vitro (Boyan, B. D., et al., "Differential expression of phenotype by resting zone and growth region costochondral chondrocytes in vitro," Bone (1988) 9:185-194; Boyan, B. D., et al., J. Biol. Chem. (1989) 264:11879-11886; Ecarot-Charrier, B., et al., Bone (1988) 9:147-154), are found in the extracellular matrix, and are associated with modification of the extracellular matrix prior to calcification.
Matrix vesicles have a distinctive phospholipid composition and enzyme activity. Their characteristics are cell-maturation dependent. Regulation of matrix vesicle structure and function occurs at the genomic and non-genomic levels. By following alkaline phosphatase gene transcription, protein concentration, and enzyme specific activity, it has been shown that steroid hormones and growth factors exhibit a regulatory influence over gene transcription, protein synthesis, and matrix vesicle activity. Matrix vesicles respond to peptide hormones such as testosterone (Schwartz, Z., et al. "Gender-specific, maturation-dependent effects of testosterone on chondrocytes in culture," Endocrinology (1994) 134:1640-1647); estrogen (Nasatzky, E., et al., "Sex-dependent effects of 17-beta-estradiol on chondrocyte differentiation in culture," J. Cell Physiol. (1993) 154:359-367); growth factors such as TGF.beta. (Bonewald, L. F., et al., "Stimulation of plasma membrane and matrix vesicle enzyme activity by transforming growth factor-beta in osteosarcoma cell cultures," J. Cell Physiol (1990) 145:200-206); other matrix proteins, like alpha 2-HS-glycoprotein (Yang, F. et al., "Alpha 2-HS-glycoprotein: expression in chondrocytes and augmentation of alkaline phosphatase and phospholipase A2 activity," Bone (1991) 12:7-15); and autocoid mediators like prostaglandins as well. Calcifying cells can modulate events in the matrix via direct autocrine/paracrine stimulation or inhibition of the matrix vesicles. 1,25-dihydroxy vitamin D.sub.3 (1,25-(OH).sub.2 D.sub.3) and 24,25-dihydroxy vitamin D.sub.3 (24,25-(OH).sub.2 D.sub.3) regulate matrix vesicle phospholipase A.sub.2 activity, fatty acid turnover, arachidonic acid release, PGE2 production, and membrane fluidity, which can act on the matrix vesicle to alter enzyme activity (Boyan, B. D., et al., "Cell maturation-specific autocrine/paracrine regulation of matrix vesicles," Bone Miner. (May 1992) 17(2):263-268).
Matrix vesicle structure and function, as well as extracellular matrix synthesis by osteoblasts and chondrocytes, are regulated by TGF.beta. as well as vitamin D metabolites (Schwartz, Z., et al., Endocrinology (1993) 132:1544-1552; Miyazono, K., et al., J. Biol. Chem. (1988) 263:6407-6415; Bonewald, L., et al., J. Cell Physiol. (1990) 145:200-206; Boyan, B. D., et al., "In vitro studies on the regulation of endochondral ossification by vitamin D," Crit. Rev. Oral Biol. Med. (1992) 3(1/2):15-30; Schwartz, Z., et al., Endocrinology (1988) 123:2878-2884; Boyan, B. D. et al., "Matrix vesicles as a marker of endochondral ossification," Connect. Tissue Res. (1990) 24:67-75; Bonewald, L. F. et al., "Stimulation of matrix vesicle enzyme activity in osteoblast-like cells by 1,25-(OH).sub.2 D.sub.3 and transforming growth factor beta (TGF beta)," Bone Miner. (1992) 17:139-144); Swain, L. D. et al., "Regulation of matrix vesicle phospholipid metabolism is cell maturation-dependent," Bone Miner. (1992) 17:192-196). Moreover, it appears that these two regulators interact in a specific manner during cell differentiation. The details of this interaction have been partially elucidated by using chondrocytes derived from costochondral cartilage. Resting zone and growth zone chondrocytes constitutively produce 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3, and TGF.beta. regulates this production (Schwartz, Z., et al., Endocrinology (1993) 132:1544-1552). Vitamin D metabolites alter membrane fluidity (Swain, L. D., et al., "Nongenomic regulation of chondrocyte membrane fluidity by 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 is dependent on cell maturation," Bone (1993) 14:609-617) and enzyme activity (Schwartz, Z. and Boyan, B., Endocrinology (1988) 122:2191-2198) of isolated matrix vesicles in vitro. Nongenomic effects of 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 have been reported and include alterations in arachidonic acid turnover (Schwartz, Z., et al., "Regulation of arachidonic acid turnover by 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 in growth zone and resting zone chondrocyte cultures," Biochim. Biophys. Acta (1990) 102:278-286; Swain, L., et al., Biochim. Biophys. Acta (1992) 1136:45-51; Boyan, B. et al., Connect. Tissue Res. (1989) 22:3-16), calcium ion flux (Langston, G. G., et al., Calcif. Tissue Int. (1990) 17:230-236; Schwartz, Z. et al., "Inhibition of 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 -dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures," J. Bone Min. Res. (1991) 6:709-718), and protein kinase C activity (Sylvia, V. L., et al., "Maturation-dependent regulation of protein kinase C activity by vitamin D.sub.3 metabolites in chondrocyte cultures," J. Cell Physiol. (1993) 157:271-278).
TGF.beta. and vitamin D have been shown to synergize with respect to alkaline phosphatase induction in bone cell lines (Bonewald, L. F., et al., Mol. Endocrinol. (1991) 5:741-751; Bonewald, L. F., et al., "Effects of combining transforming growth factor beta and 1,25-dihydroxyvitamin D.sub.3 on differentiation of a human osteosarcoma (MG-63)," J. Biol. Chem. (1992) 267:8943-8949), primary human bone cells (Wegedahl, J. E., et al., Metabolism (1992) 41:42-48), and rat resting zone chondrocytes (Schwartz, Z., et al., Endocrinology (1993) 132:1544-1552). Both TGF.beta. and vitamin D regulate chondrocyte differentiation. Exogenous TGF.beta. stimulates DNA synthesis and matrix formation in chick growth plate chondrocytes (Rosier, R. N., et al., Calcif. Tissue Res. (1988) 20:295-301; Crabb, I. D., et al., J. Bone Min. Res. (1990) 5:1105-1112; O'Keefe, R., et al., J. Bone Min. Res. (1988) 3:S67). In rat growth plate chondrocytes, rhTGF.beta.1 regulates alkaline phosphatase, phospholipase A.sub.2 (Schwartz, Z., et al., Endocrinology (1993) 132:1544-1552), as well as vitamin D metabolite production (Schwartz, Z., et al., Endocrinology (1992) 130:2495-2504). Cellular response to TGF.beta. depends on the state of endochondral maturation, with resting zone cells exhibiting a differential response compared to that observed in growth zone cell cultures. Similarly, vitamin D metabolites also regulate the expression of alkaline phosphatase (Schwartz, Z. and Boyan, B., Endocrinology (1988) 122:2191-2198), phospholipase A.sub.2, and protein kinase C (Sylvia, V. L., et al., "Maturation-dependent regulation of protein kinase C activity by vitamin D.sub.3 metabolites in chondrocyte cultures," J. Cell Physiol. (1993) 157:271-278) in chondrocytes in a cell maturation-specific manner.
Active metalloproteinases are present in matrix vesicles (Hirschman, A., et al., Calcif. Tissue Int. (1983) 35:791-797; Einhorn, T. A., et al., J. Orthop. Res. (1989) 7:792-805; Dean, D. D., et al., Calcif. Tissue Int. (1992) 50:342-349). In growth plate, the immunohistochemical distribution of TGF.beta.1 (Jingushi, S., et al., Calcium Regulation and Bone Metabolism, Cohn, D. V., Glorieux, F. H., and Martin, T. J. (eds.), Elsevier Science Publishers (Biomedical Division) New York, (1990) Vol. 10,298-303) coincides with the localization of matrix vesicles in the territorial matrix of the cells (Anderson, H. C., J. Cell Biol. (1969) 41:59-72). Active acid and neutral metalloproteinases, as well as plasminogen activator, are present in matrix vesicles and require physical destruction of the matrix vesicle membrane for their release (Dean, D. D., et al., Calcif. Tissue Int. (1992) 50:342-349).
Other enzymes present in matrix vesicles are sensitive to regulation by TGF.beta. and vitamin D metabolites (Schwartz, Z., et al., Endocrinology (1993) 132:1544-1552; Schwartz, Z., et al., Endocrinology (1988) 123:2878-2884; Sylvia, V. L., et al., J. Cell Physiol. (1993) 157:271-278; Boyan, B. D., et al., Endocrinology (1988) 122:2851-2860). In both instances the effects are cell maturation-dependent and vitamin D metabolite-specific. 1,25-(OH).sub.2 D.sub.3 stimulates matrix vesicle phospholipase A.sub.2 (Schwartz, Z. and Boyan, B., Endocrinology (1988) 122:2191-2198), increasing the production of lyso derivatives, resulting in loss of membrane integrity (Ginsburg, L. et al., Inflammation (1992) 16:519-538). In contrast, 24,25-(OH).sub.2 D.sub.3 inhibits matrix vesicle phospholipase A.sub.2 (Schwartz, Z. and Boyan, B., Endocrinology (1988) 122:2191-2198), potentially resulting in a more stable membrane and retention of metalloproteinases within the matrix vesicle.
Matrix vesicle membrane fluidity (Swain, L. D., et al., Bone (1993) 14:609-617) and enzyme activity (Schwartz, Z., et al., Endocrinology (1988) 123:2878-2884) can be directly and specifically regulated by 1,25-(OH).sub.2 D.sub.3 in the absence of the cell and its molecular and protein synthetic machinery.
Matrix vesicles have been associated with wound healing (Schmitz, J. et al., Acta Anatomica (1990) 138:185-192; Einhorn, T. A. et al., J. Orthop. Res. (1989) 7:792-805; Brighton, C. T. and Hunt, R. M., Clin. Orth. Rel. Res. (1974) 100:406-416), however the role of matrix vesicles in wound healing has not previously been known. Endochondral wound healing is stimulated by application of electrical energy possibly through stimulation of matrix vesicle production by cells. C. T. Brighton and R. M. Hunt noted that stimulation of non-union tissue with electromagnetic fields causes an increase in the number of matrix vesicles as well as in the formation of crystals and calcification of the matrix. This was followed by healing of the nonunion defect with calcified cartilage and bone.
Cartilage and bone wound healing are also aided through placing implants made of bioerodible polymers into the defects. Such bioerodible polymers are described, e.g. in U.S. patent application Ser. No. 08/123,812 filed Sep. 20, 1993, and corresponding PCT publication WO/9315694, published Aug. 19, 1993, and U.S. Pat. No. 08/196,970 filed Feb. 15, 1994, all of which are incorporated herein by reference. Such implants may contain growth factors and other agents for promotion of wound healing.
Bone-bonding implants such as KG Cera, Mina 13, and titanium support an increase in matrix vesicle concentration compared with nonbone-bonding implants (Schwartz, Z. et al., "Effect of glass ceramic and titanium implants on primary calcification during rat fibial bone healing," Calcif. Tissue Int. (1991) 49:359-364) and also lead to increased alkaline phosphatase and phospholipase A.sub.2 (Schwartz, Z. et al., "In vivo regulation of matrix vesicle concentration and enzyme activity during primary bone formation," Bone Miner. (1992) 17:134-138; Schwartz, Z. et al., "Modulation of matrix vesicle enzyme activity and phosphatidylserine content by ceramic implant materials during endosteal bone healing," Calcif. Tissue (1992) 51:429-437). Hydroxyapatite implants behave like bone-bonding implants in that there is a stimulation of matrix vesicle enzymes, increased phosphatidylserine content and increased numbers of matrix vesicles (Schwartz, Z. et al., "Effects of hydroxyapatite implants on primary mineralization during rat fibial healing: biochemical and morphometric analysis," J. Biomed. Mater. Res. 27:1029-1038).
Biodegradable polymeric scaffold systems seeded with cells are useful for culture of specific types of cells in vitro. U.S. Pat. No. 4,963,489 to Naughton et al. issued Oct. 16, 1990 for "Three-Dimensional Cell and Tissue Culture System," incorporated herein by reference, discloses the use of a polymeric matrix for culture of cells such as skin, liver, pancreas, bone marrow, osteoblasts and chondrocytes, etc. in vitro. The seeded matrix may be transplanted in vivo. Related U.S. Pat. No. 5,032,508 to Naughton et al. for "Three-Dimensional Cell and Tissue Culture System," also incorporated herein by reference, contains a similar disclosure. A further related U.S. Pat. No. 5,160,490 to Naughton et al. issued Nov. 3, 1992 for "Three-Dimensional Cell and Tissue Culture Apparatus," incorporated herein by reference, discloses that hip prostheses coated with three-dimensional cultures of cartilage may be implanted into patients. This patent also discloses that proteins can be "added to" the matrix or coated on.